Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma (original) (raw)
SULF2 protein is expressed in 50% of human GBM. By in silico analysis of human expression data (49), we found elevated expression of SULF2 in GBM (Figure 1A). Using a stringent cutoff of a 10-fold increase in SULF2 SAGE tags over levels in normal brain to define high SULF2 levels, 7 of 16 GBMs (including both primary and xenograft tumors) had increased SULF2 expression. In contrast, SULF1 expression was not altered in most tumors (Figure 1B). Strikingly, in an independent set of 424 primary human GBM tumors, SULF2 expression was increased in 46% (197/424) of tumors relative to normal brain (Figure 1C). Consistent with the expression data, we found robust expression of SULF2 protein in 4 of 6 human high-grade astrocytoma cell lines (Figure 1D). Furthermore, immunohistochemistry on an independent set of 57 primary human GBM tumors demonstrated SULF2 protein in tumor cells in 50% of tumors (29/57 tumors) (Figure 1E). In a majority of the tumors, SULF2-positive tumor cells were widely distributed throughout the tumors and many coexpressed OLIG2 (Figure 1, F–I).
SULF2 expression in human GBM. (A and B) In silico analysis of SULF2 and SULF1 expression in 16 human GBM tumor samples (49). Each bar represents normalized expression (y axis), as number of SAGE tags per million tags, for each patient tumor listed on the x axis. Expression in normal (Nl) brain is shown in each graph. (C) Increased SULF2 expression in 197/424 (46%) primary GBM tumors, log2(tumor/normal) greater than 1.0 (fold change of tumor versus normal greater than or equal to 2.0). See also Supplemental Figure 1 and Supplemental Table 1. (D) Western blot analysis of 6 human high-grade astrocytoma cell lines for SULF2 (~135 kDa). 293T cells with or without expression of mSULF2 were used as positive (+) and negative (–) controls. (E) Distribution of SULF2 protein expression in 57 primary human GBM tumors by immunohistochemistry. The percentage of SULF2-positive tumor cells was scored from no positive cells (score 0) to more than 75% of tumor cells positive (score 3) (see Methods). (F–I) Representative images from 2 SULF2-positive tumors (F, G, and I) and a SULF2-negative tumor (H). SULF2-positive (brown) tumor cells (F) were widely distributed except in occasional tumors that displayed a more prominent perivascular distribution (G). Many SULF2-positive (brown) tumor cells were also OLIG2-positive (red). See also Supplemental Figure 2. (I) Examples of SULF2-positive tumor cells (arrowheads) and microvascular proliferation characteristic of GBM (arrow). Scale bars: 50 μm (F–H); 10 μm (I).
The SULF2 gene resides within a region of chromosomal amplification in GBM on chromosome 20q13 (50). In 18 of 372 primary human GBM tumors analyzed, the region containing SULF2 was amplified, and in 12 of these tumors (67%), there was increased SULF2 expression (Supplemental Figure 1A and Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI58215DS1). Furthermore, in the human glioma cell lines, the line with the most abundant SULF2 protein also had amplification of SULF2 (Supplemental Table 2). We found no uniform trend between expression levels of SULF2 and TP53 or WT1, 2 transcription factors implicated in regulating SULF2 (51, 52) in glioma cell lines and primary human GBMs (Supplemental Figure 1, B–E).
SULF2 confers a growth advantage to human GBM cells. We transduced U251 cells, which contain moderate levels of SULF2 protein (Figure 1D), with either 1 of 2 validated SULF2 shRNAs (43) or a scrambled control shRNA (Figure 2A). Transduced cells also expressed EGFP, allowing for enrichment by flow cytometry. Over time in culture, however, the SULF2-positive cells outgrew the SULF2-knockdown cells, as reflected by a decrease in the ratio of EGFP-positive to total cells (Figure 2B). Selective growth of EGFP-negative cells was not observed in scrambled control shRNA cultures. Thus, cells were sorted for EGFP and used immediately for in vitro and in vivo assays. SULF2 knockdown resulted in a significant decrease in cell viability (Figure 2, C and D). This decrease was largely rescued by overexpression of murine Sulf2 (mSulf2) (Figure 2, E and F), as demonstrated by increased SULF2 protein (Figure 2E). A similar decrease in cell viability with SULF2 knockdown was observed in SF295 and SNB75 cells (Supplemental Figure 7, A and B).
SULF2 confers a growth advantage to human GBM cells in vitro and in vivo. (A) Knockdown of SULF2 in U251 cells by 2 different shRNA constructs; SULF2 is decreased by 83% with shRNA SULF2-A and 55% with SULF2-B compared with the scrambled shRNA control. (B) In vitro growth of EGFP-positive SULF2-A shRNA (shSULF2-A) and scrambled shRNA control (shControl) cells demonstrated a selective growth advantage of SULF2-expressing cells over cells with SULF2 knockdown, as demonstrated by a decreased ratio of GFP-positive to total cells over time. *P < 0.01; n = 3. (C and D) Decreased growth and cell viability of SULF2-A shRNA cells versus scrambled shRNA control cells, as determined by counting live cells over time (*P < 0.00005; n = 3) (C) and by the colorimetric MTT viability assay, viable cell number normalized to control, day 5 after plating (*P < 0.00005; n = 5 independent experiments) (D). (E) Overexpression of mSulf2 in cells with SULF2 knockdown and in scrambled shRNA control cells. (F) Restoration of control growth with overexpression of mSulf2 in SULF2-A shRNA–containing cells (n = 3). (G) Mean tumor volume (mm3); subcutaneous flank transplant (11 days, *P < 0.05; n = 10 mice per group). (B, C, D, F, and G) Results are depicted as mean ± SEM.
Likewise, we observed a growth advantage of SULF2-positive U251 cells in vivo following subcutaneous transplant of SULF2-knockdown cells into nude mice (Figure 2G). SULF2-knockdown resulted in smaller tumor volumes in subcutaneous tumors. Decreased tumor cell growth in vitro and in vivo with SULF2 knockdown suggested a role for SULF2 in human GBM growth.
Sulf2 is expressed in a murine model for high-grade glioma. To model invasive aspects of the adult disease further, we adapted a murine model for high-grade glioma, based on the genetic manipulation of embryonic neural progenitor/stem cells (47, 48). We cultured adult neural progenitor cells from the subventricular zone (SVZ) of 11–week-old Ink4a/Arf–/– mice, transduced them with EGFRvIII (EGFR*), a constitutively active variant of EGFR derived from a human GBM (53), cultured them as tumorigenic neurospheres, and transplanted these cells orthotopically into host mice (Figure 3A). Both of these genetic alterations are common in adult human GBM. Within 7 weeks, 100% of mice developed highly invasive high-grade glial tumors (Figure 3, B–D).
Sulf2 expression in a murine model for high-grade glioma. (A) Schema for generating high-grade, invasive glioma from adult neural progenitor cells. (B–D) Invasive, high-grade tumors generated from tumorigenic neurospheres immunostained for human EGFR and H&E. Scale bars: 300 μm (B); 50 μm (C and D). Similar to human GBM, in B, the tumor invades across the corpus callosum. (E and F) Tumor cells are hEGFR positive and exhibit robust Sulf2 expression by in situ hybridization (ISH) for the Sulf2 transcript. Scale bars: 200 μm. (G) Expression of Sulf2 protein (brown) in Olig2-positive (red) tumor cells in a primary murine tumor (arrow). Scale bar: 50 μm. See also Supplemental Figures 2 and 3.
We collected the tumors at the time of sacrifice for biochemical analysis or we cultured the tumor cells as tumor neurospheres (tumor-NS), as this enriches for tumor-initiating cells and retains the molecular properties of the parental tumor (54). Indeed, we observed that the tumorigenicity of tumor-NS increased as compared with the parental tumorigenic neurospheres, as reflected by a 38% decrease in median survival following orthotopic transplant of tumor-NS (median survival 23 days vs. 37 days; P < 0.0001).
Similar to human GBM, Sulf2 expression was readily detected in the murine tumor cells in vivo by in situ hybridization and immunohistochemistry (Figure 3, E–G, and Supplemental Figure 2). Tumor cells were identified by morphology and expression of hEGFR. Sulf2 protein was present in a subpopulation of the tumor cells including Olig2-positive cells (84.5% ± 3.2%; mean ± SEM, n = 4), occasional GFAP-positive cells, and Nestin-positive cells (Supplemental Figures 2 and 3). These data indicate that highly invasive, high-grade gliomas generated from adult neural progenitor cells have abundant expression of Sulf2 and are a useful model to study Sulf2 function.
Sulf2 confers increased tumorigenicity and proliferation. To establish Sulf2 function in glioma, we generated tumorigenic neurospheres from double-transgenic mice that were Ink4a/Arf–/– and either wild type, heterozygous, or null for Sulf2 (55). As expected, the extent of HSPG sulfation was greater in Sulf2–/– versus Sulf2+/+ neurospheres, as measured by a phage-display antibody whose binding to HSPGs depends on 6-O–sulfation (ref. 56, Supplemental Figure 4, A and B; relative MFI 2.3 versus 1.0; P < 0.05, n = 2). When tumor-NS were grown in minimal medium with only EGF and FGF2, however, Sulf2–/– and Sulf2+/+ cells had similar in vitro growth (Figure 4A). Strikingly, following orthotopic transplant, we observed a significant delay in tumor development from Sulf2–/– tumorigenic neurospheres versus those that were Sulf2+/+ or Sulf2+/–. The median survival of mice transplanted with 3 independent Sulf2–/– lines was 48 days (n = 14) compared with the median survival of mice transplanted with Sulf2+/+ cells (37 days; n = 9) or Sulf2+/– cells (38 days; n = 4; P < 0.001) (Figure 4B). In addition to prolonged mouse survival, Sulf2–/– tumors were 23% smaller than Sulf2+/+ tumors (mean ratio of tumor weight to body weight was 0.014 ± 0.00034 versus 0.018 ± 0.0014; ± SEM, P < 0.05; Supplemental Figure 4C). Although Sulf2+/+ and Sulf2–/– tumors had similar histologic appearance, only tumor-NS from Sulf2+/+ tumors expressed Sulf2 protein, and this was associated with decreased HSPG sulfation relative to Sulf2–/– tumors (Figure 4C and Supplemental Figure 4). These data support a functional requirement for Sulf2 in optimal gliomagenesis in the context of the brain microenvironment.
Prolonged survival conferred by ablation of Sulf2 in tumorigenic neurospheres. (A) Similar in vitro growth of Sulf2+/+;Ink4a/Arf–/– or Sulf2–/–;Ink4a/Arf–/– tumorigenic neurospheres when cultured under nonadherent conditions (n = 3; mean ± SEM). (B) Kaplan-Meier survival analysis. Mice transplanted with Sulf2–/– cells have prolonged survival (median survival of 48 days) relative to mice transplanted with Sulf2+/+ or Sulf2+/– cells (median survival 37 days and 38 days, respectively, P < 0.001 for Sulf2–/– [n = 14] versus Sulf2+/+ [n = 9] or Sulf2+/– [n = 4]). 3 independent Sulf2–/– tumor progenitor lines were analyzed. Censored animals (black ticks) indicate individual mice sacrificed for tumor analysis prior to signs of tumor. (C) Similar tumor histology in Sulf2+/+ and Sulf2–/– tumors (H&E) despite absence of Sulf2 protein in Sulf2–/– tumor-NS (right panels, Western blot). Sulf2–/– tumors exhibit increased sulfated HSPGs (RB4CD12) compared with Sulf2+/+ tumors. Negative control antibody (MPB49) does not bind HSPG. Scale bars: 40 μm (H&E); 10 μm (HSPG, control). (D) Kaplan-Meier survival analysis demonstrating mice transplanted with tumor-NS isolated from Sulf2–/– tumors (median survival, 35 days) retain prolonged survival relative to those with tumor-NS from Sulf2+/+ tumors (median survival, 23 days). P < 0.001 for Sulf2–/– tumor-NS (n = 11) versus Sulf2+/+ tumor-NS (n = 7). See also Supplemental Figure 4.
Because Sulf2–/– tumor progenitor cells exhibited a marked delay in tumor growth relative to controls, we wondered whether these late-forming Sulf2–/– tumors had escaped Sulf2 dependence. However, when we isolated tumor-NS from mouse tumors and retransplanted them, the Sulf2–/– cells maintained their dependence on Sulf2 and exhibited significantly (P < 0.001) delayed growth (median survival, 35 days; n = 11) relative to Sulf2+/+ cells (median survival, 23 days; n = 7) (Figure 4D), thus indicating that the growth disadvantage conferred by genetic ablation of Sulf2 is durable in vivo.
Sulf2–/– tumors had a greater than 2-fold decrease in the number of proliferating tumor cells as compared with Sulf2+/+ tumors (n = 4 for Sulf2–/– and n = 6 for Sulf2+/+; P < 0.05) (Figure 5, A–C). There was no difference in the number of tumor cells undergoing apoptosis, as determined by cleaved caspase-3 immunostaining (data not shown). These data demonstrate that ablation of Sulf2 function in vivo results in decreased tumor cell proliferation, decreased tumor growth, and prolonged survival.
Decreased tumor cell proliferation in the absence of Sulf2 in vivo. (A and B) Tumor cell proliferation, as determined by immunostaining for phospho-histone H3 (a-pH3), was greater in Sulf2+/+ tumors than in Sulf2–/– tumors. Representative pH3-positive cells indicated by arrows. Scale bars: 100 μm. Insets highlight representative positive cells. (C) Quantified proliferation data; number of mean pH3-positive cells per ×200 field per mouse in Sulf2+/+ tumors was 2-fold greater than in Sulf2–/– tumors (42.9 ± 8.7 versus 19.0 ± 4.4 [± SEM] for Sulf2+/+ [n = 4] and Sulf2–/– [n = 6], respectively). *P < 0.05. (D–I) In contrast, Sulf2+/+ and Sulf2–/– tumors exhibit similar expression of differentiation markers. Tumors, highlighted by hEGFR (D and G), express both GFAP (E and H) and Nestin (F and I). Scale bars: 60 μm. See also Supplemental Figure 5.
We found no significant differences in tumor cell morphology or differentiation between Sulf2–/– and Sulf2+/+ tumors. Both tumor types contained similar proportions of GFAP-positive and Nestin-positive tumor cells (Figure 5, D–I, and Supplemental Figure 5, A–D), and they were negative for NG2, a marker of oligodendrocyte differentiation (Supplemental Figure 5, E and F). In addition, although VEGF binds HSPG and is sensitive to SULF action (57, 58), we did not observe a phenotype resembling altered VEGF signaling (59), including differences in vascular morphology or tumor cell invasion, when we ablated Sulf2 (Supplemental Figure 5, G and H).
SULF2 alters the activity of multiple RTKs in human GBM. Since we observed a SULF2-mediated growth phenotype in brain tumor cells, we hypothesized that SULF2 may act to alter the activity of important signaling pathways in human GBM. In carcinoma, SULFs have been implicated in abnormal Wnt signaling (43–45). Wnt signaling has previously been implicated in U251 growth (60). However, we observed no SULF2-dependent increase in canonical β-catenin–dependent transcriptional activity, as detected by a TCF/LEF reporter assay (Supplemental Figure 6E).
Instead, we observed that several RTKs were influenced by knockdown of SULF2 in U251 cells (Figure 6A), with a greater than 50% reduction in the activity of 3 RTKs of significance in GBM, PDGFRα, IGF1Rβ, and EPHA2 (Figure 6B). Moreover, EGFR activity was decreased by 30%. This did not reflect a global decrease in RTK activation, but a selective decrease in the activity of specific RTKs. Indeed, the closely related PDGFRβ and EPHB2 exhibited similar degrees of phosphorylation in cells with or without SULF2 knockdown. Furthermore, FGFR3 showed increased phosphorylation with SULF2 knockdown, a finding consistent with the ability of the SULFs to decrease FGF2-mediated signaling (61).
SULF2 alters activity of several RTKs in human GBM cells. (A) RTK phosphorylation in U251 cells expressing SULF2-A shRNA or scrambled control shRNA. Individual RTKs are spotted in duplicate, and the identities of specific receptors are indicated. Positive control spots are located at the corners. See also Supplemental Figure 6. (B) Relative levels of phosphorylated RTKs in cells with knockdown of SULF2 normalized to cells with scrambled shRNA control. Duplicate spots were averaged. Data are representative of 2 independent experiments. (C) Phosphorylated and total PDGFRα levels in cells with SULF2 knockdown (S2) and scrambled shRNA control (C). Western blots were probed for GAPDH as a loading control. Results are means normalized to levels in scrambled shRNA control cells ± SEM (n = 4 independent experiments). *P < 0.01. (D) Knockdown of SULF2 decreases PDGFRα activation in response to PDGF-BB (10, 100, 200 ng/ml) stimulation. Relative levels of phosphorylated to total PDGFRα normalized to levels in unstimulated scrambled shRNA control cells. Data are representative of 2 independent experiments.
To determine whether these effects were generally true, we knocked down SULF2 in another high-grade astrocytoma cell line, SNB75, and again performed human phospho-RTK antibody arrays. As with U251 cells, SULF2 knockdown resulted in alterations in RTK activity, including decreased phosphorylation of PDGFRα and decreased cell viability (Supplemental Figure 6, A–C, and Supplemental Figure 7B). Together, these data suggest that SULF2 modulates the activity of several RTKs in GBM.
Because PDGFR is a major signaling pathway in human GBM (3, 8), we sought to validate the regulation of this RTK pathway by SULF2. Consistent with our array results, cells with knockdown of SULF2 exhibited a 43% decrease in phosphorylated PDGFRα (Figure 6C) and a modest decrease of 19% in total PDGFRα levels. Furthermore, SULF2 knockdown resulted in dramatically reduced activation of PDGFRα upon the addition of PDGF ligands (Figure 6D and Supplemental Figure 6D). Using imatinib mesylate, a RTK inhibitor with activity against PDGFRα, we demonstrated an additive decrease in both PDGFRα phosphorylation and cell survival following SULF2 knockdown (Figure 7, A and B). Similar effects were observed in SNB75 cells (Supplemental Figure 7). Overexpression of mSulf2 protein rescued the decreased activity of PDGFRα in cells with knockdown of SULF2 in an imatinib mesylate–dependent manner (Figure 7, C and D). Imatinib mesylate did not decrease the activation of EGFR (Supplemental Figure 6F).
Decreased tumor cell viability conferred by knockdown of SULF2 and inhibition of PDGFR signaling. (A) PDGFRα phosphorylation is decreased by imatinib mesylate (9 μM) in both scrambled shRNA control and SULF2-A shRNA–containing cells by Western blot. (B) Knockdown of SULF2 in combination with inhibition of PDGFRα by imatinib mesylate (9 μM) results in decreased cell viability. This effect was not observed with inhibition of EGFR signaling by AG1478 (10 μM). *P < 0.005. (C) Overexpression of mouse SULF2 (mSULF2) in control and SULF2-A shRNA–containing cells by Western blot. (D) Overexpression of mSULF2 restores PDGFRα activity in cells with knockdown of human SULF2 in an imatinib mesylate–dependent manner. All data are representative of 2 independent experiments done in quadruplicate, and data are presented as mean ± SEM. C, scrambled shRNA control; S2, SULF2-A shRNA.
Ablation of Sulf2 confers decreased activation of PDGFRα and downstream signaling pathways in murine high-grade glioma. Similar to our results in 2 human astrocytoma cell lines, Sulf2 regulated PDGFRα activity in our murine tumors. In Sulf2–/– tumor-NS, we observed a marked reduction in activation of PDGFRα, with a slight reduction in total PDGFRα levels (Figure 8, A and B). Sulf2–/– tumor-NS stimulated with PDGF-BB also had reduced activation of PDGFRα relative to Sulf2+/+ tumor-NS (Supplemental Figure 8, A and B). In contrast, there was no decrease in activation of EGFR in Sulf2–/– tumor-NS stimulated with EGF relative to Sulf2+/+ tumor-NS (Supplemental Figure 8C). Although the tumor-prone cells expressed EGFRvIII, they still responded to growth factor stimulation in vitro (Supplemental Figure 8D).
Sulf2 alters the activity of PDGFRα in murine tumor-NS. (A) Phosphorylated and total PDGFRα levels in Sulf2+/+ and Sulf2–/– tumor-NS. Western blots were probed for GAPDH as a loading control. (B) Quantification of p-PDGFRα and total PDGFRα levels in tumor-NS from Sulf2+/+ and Sulf2–/– cells normalized to mean ± SEM (n = 3 independent experiments). *P < 0.05. (C) SULF2 also affected the activity of downstream signaling pathways. Phosphorylated and total Erk1/2 (p44/p42) levels in Sulf2+/+ and Sulf2–/– tumor-NS. (D) The relative mean ratio of phosphorylated Erk to total Erk levels in Sulf2+/+ and Sulf2–/– tumor-NS normalized to Sulf2+/+ levels ± SEM (n = 3 independent experiments). *P < 0.005. (E) Sulf2+/+ tumors had more prominent phosphorylated Erk immunostaining relative to Sulf2–/– tumors. Scale bars: 50 μm.
Phosphorylation of PDGFRα results in the activation of downstream signaling pathways, including the MAPK pathway, also known to be important in human GBM. Accordingly, we observed that Sulf2–/– tumor-NS had decreased activation of the MAPK family members Erk1/2 (p44/p42) (Figure 8, C and D). Furthermore, in Sulf2+/+ tumors, we observed greater p-Erk immunostaining (2.5 ± 0.2, n = 10) than in Sulf2–/– tumors (1.6 ± 0.3, n = 11; P < 0.05) (Figure 8E). Together these data demonstrate a role for Sulf2 in modulating the activity of RTKs and downstream signaling pathways in high-grade glioma.
SULF2 expression is associated with the proneural subtype of GBM characterized by abnormalities in the PDGFRα-signaling pathway. Since we found that SULF2 alters ligand-mediated RTK activity in GBM, we hypothesized that SULF2 may be enriched in a specific molecular GBM subtype. In human tumors of different subtypes, we observed a striking difference in SULF2 expression levels using data from The Cancer Genome Atlas (TCGA) ( 9A). Interestingly, SULF2 was most highly expressed in the proneural subtype of GBM (n = 173, P < 0.005), which is characterized by alterations in PDGFRα signaling. Indeed, SULF2 expression was associated with the expression of signature genes for the proneural GBM subtype (Figure 9B; Supplemental Table 3). There was also a less robust but positive association between expression of SULF2 and genes that are characteristic of the mesenchymal GBM subtype. Consistent with SULF2 expression, immunohistochemistry for SULF2 in 28 subtyped GBMs demonstrated that proneural and mesenchymal subtypes had the most abundant SULF2 protein (Figure 9C). Interestingly, the mesenchymal subtype, including the designated signature genes, is characterized by the upregulation of genes associated with the ECM and angiogenesis (7, 8). These data suggest SULF2 may help identify functional subsets of GBM.
SULF2 expression is associated with the proneural GBM subtype. (A) SULF2 expression by human GBM subtype. Box plots show the median (range) normalized SULF2 expression levels were 8.1 (6.9–9.5), 8.9 (7.0–10.2), 7.8 (6.6–9.2), and 8.5 (7.1–9.9) for the classical, proneural, neural, and mesenchymal (mesench) subtypes, respectively (n = 173 tumors); **P < 0.005. The values within the box represent the lower quartile (Q1), median, and the upper quartile (Q3) of the distribution. The horizontal bars at the 2 ends are the smallest and largest nonoutlier observations. The circles beyond the horizontal bars represent outlying cases, defined as 1.5 times the interquartile range (Q3–Q1), below Q1 or above Q3. (B) Similarity (Pearson correlation, r) between SULF2 expression and the expression of 50 genes characterized as signature genes for each of the previously defined GBM subtypes (8). A positive coefficient denotes a positive relationship between SULF2 expression and expression of the gene of interest on the x axis (n = 202 tumors). In silico analysis for A and B was performed on expression data from the TCGA Data Portal. (C) SULF2 protein expression in primary human GBM samples of different subtypes. Tissue microarrays of previously subtyped human tumors were immunostained for SULF2 and scored. Data are represented as mean ± SEM for the classical, proneural, neural, and mesenchymal GBM subtypes, respectively (n = 28 tumors total). *P < 0.05.